1. Field of the Invention
The present invention relates generally to the field of protein production by cell culture. In particular aspects, the invention provides methods and compositions for the production of polypeptides, especially by eukaryotic cell culture, which enhance secretion and facilitate recovery of the polypeptides.
2. Description of Related Disclosures
Many eukaryotic cell surface- and secreted proteins are post-translationally processed to incorporate N-linked and O-linked carbohydrate (Kornfeld and Kornfeld (1985) Annu. Rev. Biochem. 54:631-64; Rademacher et al., (1988) Annu. Rev. Biochem. 57:785-838). Protein glycosylation is thought to subserve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion (Fieldler and Simons (1995) Cell 81:309-312; Helenius (1994) Mol. Biol. of the Cell 5:253-265; Olden et al., (1978) Cell, 13:461-473; Caton et al., (1982) Cell 37:417-427; Alexander and Elder (1984) Science 226:1328-1330; Flack et al. (1994 J. Biol. Chem. 269:14015-14020). In higher organisms, the nature and extent of glycosylation can markedly affect the circulating half-life and bio-availability of secreted proteins by mechanisms involving receptor mediated uptake and clearance (Ashwell and Morrell (1974) Adv. Enzymol. 41:99-128; Ashwell and Harford (1982) Ann. Rev. Biochem. 51:531-54). Receptor systems have been identified that are thought to play a major role in the clearance of serum proteins through recognition of various carbohydrate structures on the glycoproteins (Stockert (1995) Physiol. Rev. 75:591-609; Kery et al., (1992) Arch. Biochem. Biophys. 298:49-55). Thus, production strategies resulting in incomplete attachment of terminal sialic acid residues might provide a means of shortening the bioavailability and half-life of secreted glycoproteins by promoting clearance by the hepatic asialoglycoprotein receptor. Conversely, expression strategies resulting in saturation of terminal sialic acid attachment sites might lengthen protein bioavailability and half-life by preventing uptake by hepatic receptors. In the development of recombinant glycoproteins for use as pharmaceutical products, it has been speculated that the pharmacodynamics of recombinant proteins can be modulated by the addition or deletion of glycosylation sites from a glycoprotein""s primary structure (Berman and Lasky (1985a) Trends in Biotechnol. 3:51-53). However, studies have reported that the deletion of N-linked glycosylation sites often impairs intracellular transport and results in the intracellular accumulation of glycosylation site variants (Machamer and Rose (1988) J. Biol. Chem. 263:5955-5960; Gallagher et al., (1992) J. Virology 66:7136-7145; Collier et al., (1993) Biochemistry 32: 7818-7823; Claffey et al., (1995) Biochemica et Biophysica Acta 1246:1-9; Dube et al., (1988) J. Biol. Chem. 263:17516-17521). While glycosylation site variants of secreted proteins can be expressed intracellularly, it has proved difficult to recover useful quantities from growth conditioned cell culture medium.
It has been shown that impaired secretion of the HIV-1 envelope glycoprotein containing N-linked carbohydrate structures can be partially overcome by replacing the native signal sequence of the glycoprotein with the signal sequence and 5xe2x80x2 untranslated region of the herpes simplex virus type 1 glycoprotein D (HSV gD-1) (Lasky et al., (1986) Science 233:209-212; Berman et al., (1989) J. Virol. 63:3489-3498). Studies have reported the use of the native secretory peptide of human tissue type plasminogen activator in cell culture production of non-t-PA glycoproteins (International Publication No. WO 96/17067; Krasney and Young (1992) Cytokine 4:134-143; Rhodes et al., (1994) J. Gen. Virol. 75:207-213). None have reported a strategy for the secretion and recovery of proteins wherein one or more native glycosylation sites have been added to or deleted from the protein""s primary structure.
The present invention provides methods and compositions useful in the production of proteins by recombinant cell culture. The methods and compositions overcome intracellular retention of proteins and facilitate extracellular recovery of the produced proteins. In particular embodiments, the invention provides methods and compositions for the production of polypeptides other than tissue-type plasminogen activators (t-PA""s) utilizing a precursor peptide corresponding to the amino terminal signal and/or pro peptides naturally associated with a mammalian t-PA which act to direct the secretion of the mature t-PA. In particular embodiments, the invention provides for the export and secretion of polypeptides other than t-PA""s. For example, the invention provides methods and compositions useful in the recombinant production and extracellular recovery of novel chimeric or fusion proteins such as immunoadhesins and especially those with poor secretion kinetics. In particular embodiments the invention provides for the production and recovery of glycoproteins that have been altered from their native sequence to add or remove one or more glycosylation sites. Advantageously, the invention improves and in preferred embodiments restores the export and secretion of such glycosylation site variants. It is a further advantage of the present invention that greater quantities of the polypeptides produced utilizing the compositions and methods of the present invention can be recovered from the extracelluar medium of the cultured cells from which they are produced.
Accordingly, in particular embodiments, the invention provides nucleic acids, expression systems and host cells, as well as methods for the production of non t-PA polypeptides, such as immunoadhesins and especially glycosylation site variant polypeptides, in cell culture. In one embodiment of the present invention, a DNA construct is provided for the export and secretion of such polypeptides comprising a first DNA segment encoding a precursor peptide corresponding to all or a portion of the amino terminal signal and/or pro peptides naturally associated with a mammalian and preferably a human t-PA. The first DNA segment is operably linked, in frame, to a second DNA segment encoding the heterologous (non-t-PA) polypeptide. In particular aspects, the first DNA segment encodes a peptide corresponding to a mammalian and preferably a human t-PA pro-sequence as defined herein. According to this aspect of the present invention, the first DNA segment is operably linked to a second DNA segment encoding a heterologous polypeptide.
Certain embodiments additionally comprise a DNA segment encoding a peptide corresponding to a mammalian signal and or pro-peptide upstream of and operably linked to the first DNA segment. For example, when the heterologous polypeptide is an immunoadhesin, such as a TNFR-IgG chimera, the first DNA segment may be preceded by a DNA segment encoding a mammalian t-PA or, alternatively, a heterologous signal and or pro-sequence. According to this aspect of the invention the mammalian t-PA signal sequence or other heterologous signal and or pro-sequence and the pro-peptide amino acid sequence comprise the precursor peptide of the invention.
In preferred embodiments, the first DNA segment encodes a native or naturally occurring signal and pro peptide of a mammalian t-PA and especially a human t-PA. The second DNA sequence preferably encodes a polypeptide other than a mammalian t-PA such as, for example, an immunoadhesin. According to further aspects of the invention, the second DNA sequence encodes a naturally occurring or chimeric polypeptide other than a mammalian t-PA wherein one or more glycosylation sites have been added to or deleted from the polypeptide""s native sequence. The invention further provides a DNA construct comprising one or more additional DNA segments operably linked to the first and second DNA segments.
The invention further provides a cultured eukaryotic host cell comprising a DNA construct having a first DNA segment encoding a precursor peptide the precursor peptide corresponding to all or a portion of a mammalian t-PA signal and or pro peptide and a second DNA segment operably linked to the first DNA sequence, the second DNA sequence encoding a polypeptide other than a mammalian t-PA and preferably a polypeptide other than a mammalian t-PA wherein one or more glycosylation sites have been added to or deleted from the polypeptide.
The invention further provides methods of producing a heterologous polypeptide especially a polypeptide which has been altered to delete one or more native N-linked glycosylation sites comprising the steps of
(a) culturing a eukaryotic host cell comprising a DNA construct, the DNA construct comprising:
a first DNA segment encoding a precursor peptide corresponding to all or a portion of a mammalian tissue plasminogen activator signal and or pro peptide; and
a second DNA segment operably linked to the first DNA sequence, the second DNA sequence encoding a polypeptide other than t-PA wherein one or more native N-linked glycosylation sites of the polypeptide have been deleted from the polypeptide wherein the eukaryotic host cell expresses the first and second DNA segments and the polypeptide is secreted from the cell; and
(b) recovering the polypeptide so produced.